The role of higher-order thalamus during learning and correct performance in goal-directed behavior

Article activity feed

1. 

   Version published to 10.7554/elife.77177 on eLife

   Mar 8, 2022
2. 

   eLife

   Jun 25, 2021

   Author Response:

   Evaluation Summary:

   This study, which will be of interest to neuroscientists in the fields of learning and memory, somatosensation, and motor behavior, uses systems neuroscience tools to expand our view how the postero-medial (POm) nucleus of the thalamus contributes to goal-directed behavior. The reviewers suggested additional ontogenetic experiments to clarify the nature and specificity of those roles. They also indicated that certain alternative explanations to the experimental observations could be addressed for a more balanced presentation and interpretation of the results.

   We thank the editors and reviewers for their constructive comments. We have now performed additional analysis and revised the text which we believe has improved the manuscript.

   Reviewer #1 (Public Review):

   1. Fig 1 - Supp 1 suggests that …

   Author Response:

   Evaluation Summary:

   This study, which will be of interest to neuroscientists in the fields of learning and memory, somatosensation, and motor behavior, uses systems neuroscience tools to expand our view how the postero-medial (POm) nucleus of the thalamus contributes to goal-directed behavior. The reviewers suggested additional ontogenetic experiments to clarify the nature and specificity of those roles. They also indicated that certain alternative explanations to the experimental observations could be addressed for a more balanced presentation and interpretation of the results.

   We thank the editors and reviewers for their constructive comments. We have now performed additional analysis and revised the text which we believe has improved the manuscript.

   Reviewer #1 (Public Review):

   1. Fig 1 - Supp 1 suggests that virus expression was always limited to POm. Drawing borders expressing areas from epifluorescence images is probably very dependent on imaging parameters. The Methods indicate that the authors scaled so that no pixels were saturated. This could mean that there was some weak expression of GCaMP6f or ArchT outside of POm. As I understand it, the authors set exposure/gains by the brightest points in the image. The limited extent of the infection in the figures might just reflect its center, which is brightest, rather than its full extent. If there were GCaMP or ArchT in VPL, some results would need to be reinterpreted.

   We agree with the reviewer that the determined expression areas are dependent on imaging parameters, however, we are confident that the virus expression used for analysis in this study are confined to the POm. In this study, our analysis of targeting of POm is three-fold. First, we optimized the volume of virus loaded to the minimum necessary to observe POm projections in S1 (a single targeted injection of 60 nl). Second, we analyzed the fluorescence spread using fluorescence microscopy after every experiment. We set exposure to use the full dynamic range of the image as previously described (Gambino et al., 2014). Occasionally, the virus spread to the adjacent VPM nucleus and this was easily recognizable by the characteristic VPM projections with the barrels of the barrel cortex. These animals were excluded from this study and not further analyzed. The VPL nucleus is located further caudally in respect to the VPM and again, we were able to identify if the virus has spread to this nucleus via posthoc fluorescence microscopy. These animals were excluded from this study and not further analyzed. We note that our stereotaxic injections were not flawless and the virus occasionally spread along the injection pipette track and into high-order visual thalamic nuclei LP and LD, superficial to POm. This is shown in Figure 1. These two nuclei, however, do not target S1 (Kamishina et al., 2009; van Groen and Wyss, 1992) and were therefore not imaged within our study. Third, we analyze the projection profile in FPS1 to ensure that it corresponds to the projection profile of POm and not VPL. If there is fluorescence in non-targeted areas, then the experiments were excluded from analysis.

   An additional degree of precision is offered by our imaging and optogenetic strategy. Calcium imaging was performed in layer 1 which is targeted by POm (Meyer et al., 2010), and not VPL which targets layer 4. Therefore, spillover into VPL would not influence our imaging results as we only image axons in layer 1 which is targeted by POm. Furthermore, during the optogenetic experiments, the fiber optic was targeted to the POm (not the VPL), thus providing a secondary POm localization of the photo-inhibited region. This is now discussed in the revised manuscript.

   1. Calcium responses are weaker during the naïve state than the expert state (Fig.1D,E), similar to the start of the reversal training (Fig.4G,H). If POm encodes correct actions, why is there any response at all in naïve mice? Is that not also a sign of stimulus encoding? Might there be another correlate of correctness with regard to the task, such as an expert mouse holding their paw more firmly or still on the stimulating rod? This could alter the effective stimulus or involve different motor signals to POm.

   We agree with the reviewer that the POm is encoding the stimulus in the naïve state. This is evident in our study, and others, which show that the POm increases activity during sensory input in naïve mice. In the expert state, stimulus encoding may also be performed by a subset of POm axons, however, our findings show that, overall, there is a significant increase in the POm activity which is dependent on the behavioral performance (HIT, MISS), and not on the presentation of the stimulus. This is not due to licking motion as there was similar POm activity during the action and suppression tasks which involved licking and not licking for reward (Figure 3E). Furthermore, all experiments were monitored online via a behavioral camera to examine the location of the forepaw on the stimulus during all trials, and trials where the paw was not clearly resting on the stimulating rod were excluded from analysis. However, we cannot rule out that non-detectable changes in postures/paw grip may occur which may alter the effectiveness of the stimulus. This is now discussed in the revised manuscript.

   1. The authors are rightly concerned that licking might contribute to POm activity and expend some good effort checking this. The reversal is a good control, but doesn't produce identical POm activity. The other licking analyses, while good, did not completely rule out licking effects. First, lines 110-111 state "…as there was no correlation between licking frequency and POm axonal activity (Figure 1I)", but Fig.1I doesn't seem to support that statement. Second, the authors analyze isolated spontaneous licks, but these probably involve less licking and less overall motion than during a real response.

   We thank the reviewer for acknowledging the effort we made to assess the influence of licking behavior on POm axonal activity. We now include a more direct analysis in the revised manuscript illustrating the relationship between the licking response and POm activity. This analysis shows there is no correlation between licking and POm axonal activity (linear regression, p = 0.9228), further suggesting that POm axonal activity is not simply due to licking behavior.

   1. Many figures (Fig.1F, 2B, 3C, 4C) make it apparent that a population of axons respond very early to the stimulus itself. I understand the authors point that many of their analyses show that on average the axons are not strongly modulated by this stimulus, but this is not true of every axon. Either some of these axons are coming from cells outside of POm (see #1) or some POm cells are stimulus driven. In either case, if some axons are strongly stimulus driven, the activity of these axons will correlate with correct choices. The stimulus and correct choices are themselves highly correlated because the animals perform so well. I do not understand how stimulus encoding and choice encoding can be disentangled by either behavior or the two behaviors in comparison. Simple stimulus encoding might be further modulated by arousal or reward expectation that increases with task learning (see #6).

   In this study, we are able to disentangle stimulus encoding and choice encoding by comparing the POm axonal activity with the different behavioral performance (HIT or MISS). Here, the same stimulus is always presented (tactile, 200 Hz), however, the mouse response differs. Despite receiving the same tactile stimulus, POm signaling in forepaw S1 is significantly increased during correct HIT trials compared with MISS trials in both the action and suppression task. Therefore, we do not believe POm axonal activity is predominantly encoding sensory information in this task. We agree with the reviewer that individual POm axons are heterogenous and a subset of axons may respond to the sensory stimulus during the behavior. We now state this in the revised manuscript. However, if some axons are strongly stimulus driven, the activity of these axons should correlate with both correct and incorrect choices as the same stimulus is also delivered during MISS trials. We now highlight this in the revised manuscript.

   Simple stimulus encoding might be further modulated by arousal or reward expectation that increases with task learning. In our study, the increase in POm activity during HIT behaviour was not due to elevated task engagement as, despite similar levels of arousal (Figure 4B), POm activity in expert mice differed in comparison to chance performance (switch behaviour; Figure 4G, H). This is now discussed in detail in the revised manuscript.

   1. I was unable to understand the author's conclusion about what POm is doing. They use terms like "behavioral flexibility" to describe its purpose, but the connection of this term to POm is not explained. Is a role as a flexibility switch really supported? Why does S1 need POm to signal a correct choice? Fig.6 did not seem helpful here. Couldn't S1 just detect the stimulus on its own and transmit consequent signals to wherever they need to be to generate behavior?

   We have now revised the manuscript and clearly define behavioral flexibility and to improve the clarity of our conclusions. We believe that S1 needs POm to signal a correct choice as behavior needs to be dynamically modulated at all times. If S1 simply detected the stimulus on its own and transmitted a consequent signals to generate behavior, then important modulatory processes that lead to dynamic changes in behavior would not be processed. Along with other feedback projections, the POm targets the upper layers of the cortex, whereas external sensory information targets the layer 4 input layer. At the level of a single pyramidal neuron, this means POm input lands on the tuft dendrites whereas external sensory information lands on the proximal basal dendrites. This segregation of input provides a great cellular mechanism for increasing the computational capabilities of neurons. Since the POm is most active in the expert state during correct behavior, we believe the POm plays a vital role in providing behaviorally relevant information. Our findings illustrate that the POm is simply not conveying a ‘Go’ signal as POm activity was not increased during correct behavior in chance performance.

   1. Arousal or reward expectation may be better explanations than flexibility. Lines 323-324 say that POm activity increased with pupil diameter normally but reversed during reward delivery. Which data support this statement? With regards to pupil, the Results only seem to indicate that there is no difference in diameter between the two conditions (expert and 50% chance) using 3 bins of data. However, I could not find the time windows used for computing these. Pupil is known to be lagged and the timing could be critical.

   The statement that ‘POm activity increased with pupil diameter normally but reversed during reward delivery’ stems from data illustrated in Figure 1I and 3B. For space and flow of the manuscript, we weren’t able to show them on the same graph as per below. Here, you can see that during reward (blue), POm activity decreased compared to response (green) whereas the pupil diameter was maximum during reward delivery. We now include more information in the methods regarding pupil tracking (see line 908 to 916, Data analysis and statistical methods; Pupil tracking).

   

   1. There are other possible interpretations of the results when the authors target POm for optogenetic suppression (around lines 246-248). The effects here are also consistent with preventing tonic and evoked POm activity from reaching lots of target structures other than S1: S2, PPC, motor cortex, dorsolateral striatum, etc. Maybe one of these cannot respond to the stimulus as well and Hits decrease?

   We now include a discussion in the revised manuscript that ‘since the POm targets many cortical and subcortical regions (Alloway et al., 2017; Oh et al., 2014; Trageser and Keller, 2004; Yamawaki and Shepherd, 2015), target-specific photo-inhibition is required to illustrate which POm projection pathway specifically influences goal-directed behavior.’

   1. Line 689. What alerts the mouse that a catch trial is happening? Is there something like an audio cue for onset of stimulus trials and catch trials? If there is no cue, wouldn't mice be in a different behavioral state during catch trials than during stimulus trials? The trial types could differ by more than the presence of the stimulus.

   There is broadband noise during the trial that acts as a cue. This is detailed in the methods and text.

   Reviewer #2 (Public Review):

   In this manuscript, D LaTerra et al explored the function of POm neurons during a tactile-based, goal-directed reward behavior. They target POm neurons that project to forepaw S1 and use two-photon Ca2+imaging in S1 to monitor activity as mice performed a task where forepaw tactile stimulation (200 Hz, 500 ms) predicted a reward if mice licked at a reward port within 1.5 seconds. If mice did not lick, there was a time-out instead of a reward. The authors found that POm-S1 axons showed enhanced responses during the baseline period, the response window after the cue, and during reward delivery. They then showed that a subset of neurons were active during the response window during correct trials when the tactile stimulus served as a cue, but not on catch trials where animals spontaneously licked for a reward.

   They then showed that POm axonal activity in S1 increased during the response window for "HIT" trials where animals correctly responded to the tactile stimulus with licking but the activity was less during "MISS" trials where animals did not respond. In order to probe whether this activity in the response window was being driven by motor activity, they designed a suppression task in which animals had to learn to suppress licking in response to the tactile stimulus in order to the receive a reward. POm neurons also showed increased activity during the response window even though action was being suppressed. However, this activity was less than during the action task. Thus, although POm activity is not encoding action, its activity is significantly different during an action-based task than an action suppression one. They then analyzed calclium activity during the training period between the action task and the suppression task in which animals were learning the new contingency and were not performing as experts. In this non-expert context there was not a difference between in POm axonal activity between "HIT" and "MISS" trials.

   Lastly, they used ArchT to inhibit POm cell body activity during the tactile stimulus and response window of some trials and showed that they reduced performance during the trials when light was on.

   Altogether, this paper provides evidence that POm neurons are not simply encoding sensory information. They are modulated by learning and their activity is correlated to performance in this goal-directed task. However, the actual role of the POm input to S1 is not discernable from the current experiments. Subsets of neurons show significant activity during the response window as well as reward. In addition, the role of this input is different during the switch task than during expert performance. There are a number of outstanding questions, which, if answered, would help to directly define the role of these neurons in this specific paradigm. For instance, the authors record specifically from POm axons in S1. How distinct is this activity from other neurons in the POm? Some POm neurons still show significant activity during MISS trials. Do these neurons have a different function than those that show a preferential response during HIT trials? Does POm activity during the switch task, which has a component of extinction training, differ from when the animals are first learning the action-based task? Likewise, are the same neurons that acquire a response during the initial learning of the action-based task, the same neurons that are responding during the action suppression task?

   The authors provide great evidence that POm neurons that project to the S1 do not simply encode sensory information or actions, but are instead signaling during correct performance. However, inhibition of cell bodies did not dramatically effect performance and it is still unclear what role this circuit actually plays in this behavior. Finer-tuned optogenetic experiments and analysis of cell bodies within POm may provide greater details that will help define this circuit's role.

   We thank the reviewer for their comments. We have now revised the manuscript to clearly state the role of the POm during the goal-directed behavioral tasks used in this study. We have provided more information regarding the range of activity patterns in POm axons within S1.

   The POm contains a heterogenous population of neurons and since it projects to multiple cortical and subcortical regions, the activity of POm axonal projections in S1 may indeed be different to other projection targets.

   The activity of POm axons during MISS behavior may have a different function than those that show a preferential response during HIT trials, however, this evoked rate is not significantly different to baseline and therefore is hard to differentiate from spontaneous activity (see Figure 2). Furthermore, the evoked rate of POm activity during the switch task is not significantly different compared to naïve mice (p = 0.159; Kruskal-Wallis test). This information is now included in the manuscript.

   It is unknown whether the same neurons that acquire a response during the initial learning of the action-based task are the same neurons that are responding during the action suppression task as we were unable to conclusively determine whether or not the same POm axons were imaged in the different protocols.

   Reviewer #3 (Public Review):

   In their paper "Higher order thalamus flexibly encodes correct goal-directed behavior", LaTerra et al. investigate the function of projections from the thalamic nucleus POm to primary somatosensory cortex (S1) in the performance of goal-directed behaviors. The authors performed in vivo calcium imaging of POm axons in layer 1 of the forepaw region of S1 (fpS1) to monitor the activity of POm-fpS1 projections while mice performed a tactile detection task. They report that the activity of POm-fpS1 axons on successful ('hit') trials was increased in trained mice relative to naïve mice. Additionally, the authors used an action suppression variant of the task to show that POm-fpS1 axon activity was higher on successful trials over unsuccessful ('miss') trials regardless of the correct motor response required. During transition between task conditions, when mice perform at chance levels, the increase of POm-fpS1 activity during correct trials is no longer seen. Finally, the authors use inhibitory optogenetic tools to suppress POm activity, revealing a modest suppression in behavioral success. The authors conclude from these data that POm-fpS1 axons preferentially "encode and influence correct action selection" during tactile goal-oriented behavior.

   This study presents several interesting findings, particularly with respect to the change in activity of POm-fpS1 axons during successful execution of a trained behavior. Additionally, the similarity in responses of POm-fpS1 on both the 'goal-directed action' and 'action suppression' tasks provides convincing evidence that POm-fpS1 activity is not likely to encode the motor response. Overall, these results have important implications for how activity in higher order thalamic nuclei corresponds to learning a sensorimotor behavior, and the authors use several clever experiments to address these questions. Yet, the major claim that POm encodes 'correct performance' should be defined more clearly. As is, there are alternative explanations that could be raised and should be discussed in more depth (Points 1), especially as it relates to any causal role the authors ascribe to POm (Point 2). In addition some clarification as to which types of signals (i.e. frequency of active axons vs. amplitude of signal in the active axons) the authors feel are most informative would be helpful (Point 3).

   We thank the reviewer for their helpful comments and assessment of our study. We have now addressed all comments and revised the manuscript accordingly.

   1. The authors argue that POm activity reflects 'correct task performance' and that the increased activity of POm-fpS1 axons in the response epoch is not due to sensory encoding. An alternative explanation is that POm-fpS1 axons do convey sensory information, and these connections are facilitated with learning - meaning the activity of pathways conveying sensory signals that are correlated with task success could be facilitated with training, and this facilitation could be disrupted during the switching task. In this sense, the activity profiles do not encode 'correct action' per se, but rather represent the sensory responses whose correlation to rewarded action have been reinforced with training (which would also be a very interesting finding). This would be quite distinct from the "cognitive functions" they ascribe to this pathway (line 341). It might have helped to introduce a delay period in between the sensory stimulus and response epoch to try to distinguish responses that encode information about the sensory stimulus from those that might be involved in encoding task performance. However, as is, it is difficult to distinguish between these two scenarios with this data, and thus the interpretations the authors present could be rephrased with alternatives discussed in more depth.

   Based on multiple findings within this study, we suggest that the POm does not predominantly encode sensory information. This is most evident when comparing POm activity during correct (HIT) and incorrect (MISS) behavior in both the action and suppression tasks. As shown in Figures 2 and 3, there is a considerable difference in activity during correct (HIT) and incorrect (MISS) trials, even though the same stimulus was delivered in both trial types. This finding suggests that POm axons do not convey sensory information which is facilitated with learning as, if this were true, it could be expected that HIT and MISS responses would be similarly increased in expert (HIT and MISS) compared to naïve mice. This is now discussed in detail in the revised manuscript.

   We agree that it would have been beneficial to separate the stimulus from the response period in the behavioral paradigm. However, to avoid engaging working memory, we did not wish to enforce a delay period in this study. We have, in another study, enforced a short delay period (500 ms) between the stimulus and response epoch. Here, the evoked rate of POm axonal activity in expert mice was three-fold greater in the (now clearly separated) response epoch compared to the stimulus epoch (0.30 ± 0.02 vs. 0.099 ± 0.01, n = 196 dendrites; p < 0.0001; Wilcoxon matched-pairs signed rank test). Although out of the scope of this study, these unpublished results provides further confirmation and confidence in the analysis performed and conclusions made in this study.

   1. Similarly, while the authors attempt to establish a causal role for POm in task performance by optogenetically inhibiting POm during the response epoch, the results are also consistent with a deficit in sensory processing, and cannot be interpreted strictly as a disruption of the encoding of 'correct action' task performance signals. Furthermore, these perturbation studies do not demonstrate that the POm-fpS1 projections they are studying are implicated in the modest behavioral deficits. As the authors state, POm projects to many targets (lines 63-66), and similar sensory-based, goal-directed behaviors do not require S1 (lines 302-305). In light of these points, some of the statements ascribing a causal role for these projections in task success could be rephrased (e.g. line 33 "to encode and influence correct action selection", line 252 "a direct influence", line 340 "plays an active role during correct performance").

   We agree that the decrease in correct performance during optogenetic inhibition of POm cell bodies may also be explained by a deficit in sensory processing. However, in this study, we went to great lengths to illustrate that the POm is encoding correct action, and not sensory information (detailed in response to 1). This is further expanded upon in the revised manuscript. We also agree that the perturbation studies do not directly demonstrate that the POm to S1 projections are driving the behavioral deficits. We therefore only refer to the POm itself when discussing the influence on behavior and we have now revised the manuscript accordingly.

   1. Event amplitude and probability were both quantified, but were not consistently reported throughout the manuscript and figures. For example, Figure 1 reports both probability and amplitude (Figure 1G and H), whereas Figure 2 only reports probability. Thus, it was not always clear as to whether the authors were ascribing biological significance to one or both of these measures, given that in some cases differences were found in one and not the other, and which of the measures were reported was occasionally switched. It would be helpful for the authors to clarify the significance they assign to each measure, and report both measures side by side for all experiments if they interpret them both as relevant.

   We thank the reviewer for this observation and have now included a statement discussing the reporting of Ca2+ transient probability and/or amplitude in the methods. Throughout the Figures, we typically illustrated probability of an evoked transient as this is a reliable measure which was dramatically altered within this study. We now report the Ca2+ transient peak amplitudes during HIT and MISS trials for direct comparison of both measures (Figure 2).

   Read the original source
3. 

   eLife

   Jun 25, 2021

   Evaluation Summary:

   This study, which will be of interest to neuroscientists in the fields of learning and memory, somatosensation, and motor behavior, uses systems neuroscience tools to expand our view how the postero-medial (POm) nucleus of the thalamus contributes to goal-directed behavior. The reviewers suggested additional ontogenetic experiments to clarify the nature and specificity of those roles. They also indicated that certain alternative explanations to the experimental observations could be addressed for a more balanced presentation and interpretation of the results.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

   Read the original source
4. 

   eLife

   Jun 25, 2021

   Reviewer #1 (Public Review):

   1. Fig 1 - Supp 1 suggests that virus expression was always limited to POm. Drawing borders expressing areas from epifluorescence images is probably very dependent on imaging parameters. The Methods indicate that the authors scaled so that no pixels were saturated. This could mean that there was some weak expression of GCaMP6f or ArchT outside of POm. As I understand it, the authors set exposure/gains by the brightest points in the image. The limited extent of the infection in the figures might just reflect its center, which is brightest, rather than its full extent. If there were GCaMP or ArchT in VPL, some results would need to be reinterpreted.

   2. Calcium responses are weaker during the naïve state than the expert state (Fig.1D,E), similar to the start of the reversal training (Fig.4G,H). If POm encodes correct …

   Reviewer #1 (Public Review):

   1. Fig 1 - Supp 1 suggests that virus expression was always limited to POm. Drawing borders expressing areas from epifluorescence images is probably very dependent on imaging parameters. The Methods indicate that the authors scaled so that no pixels were saturated. This could mean that there was some weak expression of GCaMP6f or ArchT outside of POm. As I understand it, the authors set exposure/gains by the brightest points in the image. The limited extent of the infection in the figures might just reflect its center, which is brightest, rather than its full extent. If there were GCaMP or ArchT in VPL, some results would need to be reinterpreted.

   2. Calcium responses are weaker during the naïve state than the expert state (Fig.1D,E), similar to the start of the reversal training (Fig.4G,H). If POm encodes correct actions, why is there any response at all in naïve mice? Is that not also a sign of stimulus encoding? Might there be another correlate of correctness with regard to the task, such as an expert mouse holding their paw more firmly or still on the stimulating rod? This could alter the effective stimulus or involve different motor signals to POm.

   3. The authors are rightly concerned that licking might contribute to POm activity and expend some good effort checking this. The reversal is a good control, but doesn't produce identical POm activity. The other licking analyses, while good, did not completely rule out licking effects. First, lines 110-111 state "...as there was no correlation between licking frequency and POm axonal activity (Figure 1I)", but Fig.1I doesn't seem to support that statement. Second, the authors analyze isolated spontaneous licks, but these probably involve less licking and less overall motion than during a real response.

   4. Many figures (Fig.1F, 2B, 3C, 4C) make it apparent that a population of axons respond very early to the stimulus itself. I understand the authors point that many of their analyses show that on average the axons are not strongly modulated by this stimulus, but this is not true of every axon. Either some of these axons are coming from cells outside of POm (see #1) or some POm cells are stimulus driven. In either case, if some axons are strongly stimulus driven, the activity of these axons will correlate with correct choices. The stimulus and correct choices are themselves highly correlated because the animals perform so well. I do not understand how stimulus encoding and choice encoding can be disentangled by either behavior or the two behaviors in comparison. Simple stimulus encoding might be further modulated by arousal or reward expectation that increases with task learning (see #6).

   5. I was unable to understand the author's conclusion about what POm is doing. They use terms like "behavioral flexibility" to describe its purpose, but the connection of this term to POm is not explained. Is a role as a flexibility switch really supported? Why does S1 need POm to signal a correct choice? Fig.6 did not seem helpful here. Couldn't S1 just detect the stimulus on its own and transmit consequent signals to wherever they need to be to generate behavior?

   6. Arousal or reward expectation may be better explanations than flexibility. Lines 323-324 say that POm activity increased with pupil diameter normally but reversed during reward delivery. Which data support this statement? With regards to pupil, the Results only seem to indicate that there is no difference in diameter between the two conditions (expert and 50% chance) using 3 bins of data. However, I could not find the time windows used for computing these. Pupil is known to be lagged and the timing could be critical.

   7. There are other possible interpretations of the results when the authors target POm for optogenetic suppression (around lines 246-248). The effects here are also consistent with preventing tonic and evoked POm activity from reaching lots of target structures other than S1: S2, PPC, motor cortex, dorsolateral striatum, etc. Maybe one of these cannot respond to the stimulus as well and Hits decrease?

   8. Line 689. What alerts the mouse that a catch trial is happening? Is there something like an audio cue for onset of stimulus trials and catch trials? If there is no cue, wouldn't mice be in a different behavioral state during catch trials than during stimulus trials? The trial types could differ by more than the presence of the stimulus.

   Read the original source
5. 

   eLife

   Jun 25, 2021

   Reviewer #2 (Public Review):

   In this manuscript, D LaTerra et al explored the function of POm neurons during a tactile-based, goal-directed reward behavior. They target POm neurons that project to forepaw S1 and use two-photon Ca2+imaging in S1 to monitor activity as mice performed a task where forepaw tactile stimulation (200 Hz, 500 ms) predicted a reward if mice licked at a reward port within 1.5 seconds. If mice did not lick, there was a time-out instead of a reward. The authors found that POm-S1 axons showed enhanced responses during the baseline period, the response window after the cue, and during reward delivery. They then showed that a subset of neurons were active during the response window during correct trials when the tactile stimulus served as a cue, but not on catch trials where animals spontaneously licked for a reward.

   T…

   Reviewer #2 (Public Review):

   In this manuscript, D LaTerra et al explored the function of POm neurons during a tactile-based, goal-directed reward behavior. They target POm neurons that project to forepaw S1 and use two-photon Ca2+imaging in S1 to monitor activity as mice performed a task where forepaw tactile stimulation (200 Hz, 500 ms) predicted a reward if mice licked at a reward port within 1.5 seconds. If mice did not lick, there was a time-out instead of a reward. The authors found that POm-S1 axons showed enhanced responses during the baseline period, the response window after the cue, and during reward delivery. They then showed that a subset of neurons were active during the response window during correct trials when the tactile stimulus served as a cue, but not on catch trials where animals spontaneously licked for a reward.

   They then showed that POm axonal activity in S1 increased during the response window for "HIT" trials where animals correctly responded to the tactile stimulus with licking but the activity was less during "MISS" trials where animals did not respond. In order to probe whether this activity in the response window was being driven by motor activity, they designed a suppression task in which animals had to learn to suppress licking in response to the tactile stimulus in order to the receive a reward. POm neurons also showed increased activity during the response window even though action was being suppressed. However, this activity was less than during the action task. Thus, although POm activity is not encoding action, its activity is significantly different during an action-based task than an action suppression one. They then analyzed calclium activity during the training period between the action task and the suppression task in which animals were learning the new contingency and were not performing as experts. In this non-expert context there was not a difference between in POm axonal activity between "HIT" and "MISS" trials.

   Lastly, they used ArchT to inhibit POm cell body activity during the tactile stimulus and response window of some trials and showed that they reduced performance during the trials when light was on.

   Altogether, this paper provides evidence that POm neurons are not simply encoding sensory information. They are modulated by learning and their activity is correlated to performance in this goal-directed task. However, the actual role of the POm input to S1 is not discernable from the current experiments. Subsets of neurons show significant activity during the response window as well as reward. In addition, the role of this input is different during the switch task than during expert performance. There are a number of outstanding questions, which, if answered, would help to directly define the role of these neurons in this specific paradigm. For instance, the authors record specifically from POm axons in S1. How distinct is this activity from other neurons in the POm? Some POm neurons still show significant activity during MISS trials. Do these neurons have a different function than those that show a preferential response during HIT trials? Does POm activity during the switch task, which has a component of extinction training, differ from when the animals are first learning the action-based task? Likewise, are the same neurons that acquire a response during the initial learning of the action-based task, the same neurons that are responding during the action suppression task?

   The authors provide great evidence that POm neurons that project to the S1 do not simply encode sensory information or actions, but are instead signaling during correct performance. However, inhibition of cell bodies did not dramatically effect performance and it is still unclear what role this circuit actually plays in this behavior. Finer-tuned optogenetic experiments and analysis of cell bodies within POm may provide greater details that will help define this circuit's role.

   Read the original source
6. 

   eLife

   Jun 25, 2021

   Reviewer #3 (Public Review):

   In their paper "Higher order thalamus flexibly encodes correct goal-directed behavior", LaTerra et al. investigate the function of projections from the thalamic nucleus POm to primary somatosensory cortex (S1) in the performance of goal-directed behaviors. The authors performed in vivo calcium imaging of POm axons in layer 1 of the forepaw region of S1 (fpS1) to monitor the activity of POm-fpS1 projections while mice performed a tactile detection task. They report that the activity of POm-fpS1 axons on successful ('hit') trials was increased in trained mice relative to naïve mice. Additionally, the authors used an action suppression variant of the task to show that POm-fpS1 axon activity was higher on successful trials over unsuccessful ('miss') trials regardless of the correct motor response required. …

   Reviewer #3 (Public Review):

   In their paper "Higher order thalamus flexibly encodes correct goal-directed behavior", LaTerra et al. investigate the function of projections from the thalamic nucleus POm to primary somatosensory cortex (S1) in the performance of goal-directed behaviors. The authors performed in vivo calcium imaging of POm axons in layer 1 of the forepaw region of S1 (fpS1) to monitor the activity of POm-fpS1 projections while mice performed a tactile detection task. They report that the activity of POm-fpS1 axons on successful ('hit') trials was increased in trained mice relative to naïve mice. Additionally, the authors used an action suppression variant of the task to show that POm-fpS1 axon activity was higher on successful trials over unsuccessful ('miss') trials regardless of the correct motor response required. During transition between task conditions, when mice perform at chance levels, the increase of POm-fpS1 activity during correct trials is no longer seen. Finally, the authors use inhibitory optogenetic tools to suppress POm activity, revealing a modest suppression in behavioral success. The authors conclude from these data that POm-fpS1 axons preferentially "encode and influence correct action selection" during tactile goal-oriented behavior.

   This study presents several interesting findings, particularly with respect to the change in activity of POm-fpS1 axons during successful execution of a trained behavior. Additionally, the similarity in responses of POm-fpS1 on both the 'goal-directed action' and 'action suppression' tasks provides convincing evidence that POm-fpS1 activity is not likely to encode the motor response. Overall, these results have important implications for how activity in higher order thalamic nuclei corresponds to learning a sensorimotor behavior, and the authors use several clever experiments to address these questions. Yet, the major claim that POm encodes 'correct performance' should be defined more clearly. As is, there are alternative explanations that could be raised and should be discussed in more depth (Points 1), especially as it relates to any causal role the authors ascribe to POm (Point 2). In addition some clarification as to which types of signals (i.e. frequency of active axons vs. amplitude of signal in the active axons) the authors feel are most informative would be helpful (Point 3).

   1. The authors argue that POm activity reflects 'correct task performance' and that the increased activity of POm-fpS1 axons in the response epoch is not due to sensory encoding. An alternative explanation is that POm-fpS1 axons do convey sensory information, and these connections are facilitated with learning - meaning the activity of pathways conveying sensory signals that are correlated with task success could be facilitated with training, and this facilitation could be disrupted during the switching task. In this sense, the activity profiles do not encode 'correct action' per se, but rather represent the sensory responses whose correlation to rewarded action have been reinforced with training (which would also be a very interesting finding). This would be quite distinct from the "cognitive functions" they ascribe to this pathway (line 341). It might have helped to introduce a delay period in between the sensory stimulus and response epoch to try to distinguish responses that encode information about the sensory stimulus from those that might be involved in encoding task performance. However, as is, it is difficult to distinguish between these two scenarios with this data, and thus the interpretations the authors present could be rephrased with alternatives discussed in more depth.

   2. Similarly, while the authors attempt to establish a causal role for POm in task performance by optogenetically inhibiting POm during the response epoch, the results are also consistent with a deficit in sensory processing, and cannot be interpreted strictly as a disruption of the encoding of 'correct action' task performance signals. Furthermore, these perturbation studies do not demonstrate that the POm-fpS1 projections they are studying are implicated in the modest behavioral deficits. As the authors state, POm projects to many targets (lines 63-66), and similar sensory-based, goal-directed behaviors do not require S1 (lines 302-305). In light of these points, some of the statements ascribing a causal role for these projections in task success could be rephrased (e.g. line 33 "to encode and influence correct action selection", line 252 "a direct influence", line 340 "plays an active role during correct performance").

   3. Event amplitude and probability were both quantified, but were not consistently reported throughout the manuscript and figures. For example, Figure 1 reports both probability and amplitude (Figure 1G and H), whereas Figure 2 only reports probability. Thus, it was not always clear as to whether the authors were ascribing biological significance to one or both of these measures, given that in some cases differences were found in one and not the other, and which of the measures were reported was occasionally switched. It would be helpful for the authors to clarify the significance they assign to each measure, and report both measures side by side for all experiments if they interpret them both as relevant.

   Read the original source
7. 

   Version published to 10.1101/2020.07.05.188821 on bioRxiv

   Jul 6, 2020